Divergent and convergent synthesis of polymannosylated dibranched antigenic peptide of the immunodominant epitope MBP(83–99)

Multiple antigenic peptide (MAP) systems are dendrimeric structures bearing multiple copies of identical or different peptide epitopes, and they have been demonstrated to show enhanced immunogenicity. Herein, we report the direct (divergent) and indirect (convergent) synthesis, using contemporary synthetic approaches, of a di-branched antigenic peptide (di-BAP) containing the immunodominant epitope MBP(83–99), which is implicated in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). The direct synthesis (di-BAP 1) was performed using microwave irradiation. The indirect synthesis (di-BAP 2) was carried out performing an efficient chemoselective coupling reaction through the formation of a thioether bond. Both di-BAPs were conjugated to polysaccharide mannan since mannosylation is a promising technique to achieve modulation in immune response. The conjugation was achieved through free amino groups of both di-BAPs via the formation of Schiff bases. The mannan-conjugated di-BAPs were further evaluated in vivo in a prophylactic vaccination protocol, prior to EAE induction in Lewis rats.     

Synthesis of lipopeptides of the immunodominant epitope hMBP(83–99) containing amide or C-C bond linked hydrophobic chains for the study of T cell response

Summary We previously demonstrated that the lipopeptide of the myelin basic protein (MBP) immunodominant epitope in Lewis rat Palm-GpMBP(74-85) (Gp: guinea pig), which induced experimental autoimmune encephalomyelitisin vivo strongly increased the T cell proliferative responsein vitro. We extended this study to the human immunodominant epitope hMBP(83-99), synthesizing different lipophilic peptides bearing a hydrophobic chain linked through an amide or a C-C bond. To this aim, we developed a synthetic pathway for (±)-N-Fmoc-Ahd-OH (Ahd: 2-aminohexadecanoic acid) which was used to synthesize diastereomeric peptides which were successfully separated by reverse-phase high-performance liquid chromatography. MBP-specific T cell lines recognizing the immunodominant epitope hMBP(83-99) have been generated from patients affected by multiple sclerosis. Their proliferative response to the native peptide and to some lipoderivatives has been investigated. In contrast to the animal model, none of the investigated lipopeptides exhibited ‘superagonist’ activity. Prof. L. Amaducci passed away on 11 January 1998. His memory will hearten those pursuing this research.     

Synthesis of lipopeptides of the immunodominant epitope hMBP(83–99) containing amide or C-C bond linked hydrophobic chains for the study of T cell response

We previously demonstrated that the lipopeptide of the myelin basic protein (MBP) immunodominant epitope in Lewis rat Palm-GpMBP(74–85) (Gp: guinea pig), which induced experimental autoimmune encephalomyelitis in vivo strongly increased the T cell proliferative response in vitro. We extended this study to the human immunodominant epitope hMBP(83–99), synthesizing different lipophilic peptides bearing a hydrophobic chain linked through an amide or a C-C bond. To this aim, we developed a synthetic pathway for (±)-N-Fmoc-Ahd-OH (Ahd: 2-aminohexadecanoic acid) which was used to synthesize diastereomeric peptides which were successfully separated by reverse-phase high-performance liquid chromatography. MBP-specific T cell lines recognizing the immunodominant epitope hMBP(83–99) have been generated from patients affected by multiple sclerosis. Their proliferative response to the native peptide and to some lipoderivatives has been investigated. In contrast to the animal model, none of the investigated lipopeptides exhibited superagonist activity.     

High-throughput docking for the identification of new influenza A virus polymerase inhibitors targeting the PA–PB1 protein–protein interaction

A high-throughput molecular docking approach was successfully applied for the selection of potential inhibitors of the Influenza RNA-polymerase which act by targeting the PA–PB1 protein–protein interaction. Commercially available compounds were purchased and biologically evaluated in vitro using an ELISA-based assay. As a result, some compounds possessing a 3-cyano-4,6-diphenyl-pyridine nucleus emerged as effective inhibitors with the best ones showing IC₅₀ values in the micromolar range.     

Peptide immunisation of HLA-DR–transgenic mice permits the identification of a novel HLA-DRβ1*0101– and HLA-DRβ1*0401–restricted epitope from p53

Because of the central role of CD4⁺ T cells in antitumour immunity, the identification of the MHC class II–restricted peptides to which CD4⁺ T cells respond has become a priority of tumour immunologists. Here, we describe a strategy permitting us to rapidly determine the immunogenicity of candidate HLA-DR–restricted peptides using peptide immunisation of HLA-DR–transgenic mice, followed by assessment of the response in vitro. This strategy was successfully applied to the reported haemaglutinin influenza peptide HA(307–319), and then extended to three candidate HLA-DR–restricted p53 peptides predicted by the evidence-based algorithm SYFPEITHI to bind to HLA-DR1*0101 (HLA-DR1) and HLA-DR1*0401 (HLA-DR4) molecules. One of these peptides, p53(108–122), consistently induced responses in HLA-DR1– and in HLA-DR4–transgenic mice. Moreover, this peptide was naturally processed by dendritic cells (DCs), and induced specific proliferation in the splenocytes of mice immunised with p53 cDNA, demonstrating that immune responses could be naturally mounted to the peptide. Furthermore, p53(108–122) peptide was also immunogenic in HLA-DR1 and HLA-DR4 healthy donors. Thus, the use of this transgenic model permitted the identification of a novel HLA-DR–restricted epitope from p53 and constitutes an attractive approach for the rapid identification of novel immunogenic MHC class II–restricted peptides from tumour antigens, which can ultimately be incorporated in immunotherapeutic protocols.     

Gas chromatographic–mass spectrometry and gas chromatographic–Fourier transform infrared spectroscopy assay for the simultaneous identification of fentanyl metabolites

Fentanyl, a synthetic opioid, undergoes important biotransformation to several metabolites. A gas chromatographic–mass spectrometric assay was applied for the simultaneous analysis of fentanyl and its major metabolites in biological samples. The identification of different metabolites was performed by gas chromatography–mass spectrometry (electronic impact and chemical ionisation modes) and gas chromatography–Fourier transform infrared spectroscopy. In the present study, rat and human microsomes incubation mixtures and human urines were analysed. In vitro formation of already known fentanyl metabolites was confirmed. The presence of metabolites not previously detected in human urine is described.     

I point my heart with the tip of my fingers–Biometry for the diagnosis of congenital heart defects

Congenital heart defects are characterized by abnormal positioning of some anatomical structures relative to a normal heart. Classically the classification of the disease or the stage of the disease is based on the measurement of the relative position of these structures by the cardiac morphologist. We propose a method in which the heart volume is acquired by a CT scanner. In this paper we present a tool which allows us to define landmarks interactively in this heart volume. These landmarks are then used to estimate some simple geometrical models of the anatomical structures and so as to describe their relative position and orientation.     

Validation of a one-step PCR assay for the molecular identification of Echinococcus granulosus sensu stricto G1–G3 genotype

Molecular Biology Reports - The Italian National Reference Center for Echinococcosis (CeNRE, Sassari, Italy) set up a diagnostic protocol of “one-step-PCR” useful for the detection of...     

Electrophoretic protein analysis for the identification of doubled haploid 1A–1R, 1B–1R wheat-rye double translocation lines and for the assessment of their genetic stability

Eighteen available doubled haploid wheat lines with a cytologically proven 1A–1R, 1B–1R double translocation, which where derived via anther culture from four crosses of the 1A–1R wheat-rye translocation cv Amigo with several 1B–1R wheat-rye translocation forms, were subjected to electrophoretic seed protein analysis. Besides, the five parents used in the crosses and some other wheat cultivars and doubled haploid lines (19 with a 1B–1R single translocation, 10 with a 1A–1R translocation and 7 without any 1R translocation) were also included in the investigation. It was found that the gliadin patterns visualized after SDS polyacrylamide gel electrophoresis of alcohol-soluble seed protein extracts can differentiate not only 1B–1R and 1A–1R translocation forms from wheats without any 1R-translocation chromosome, but also 1B–1R and 1A–1R wheats from each other. Moreover, 1A–1R, 1B–1R double translocation lines can be distinguished as well due to characteristic differences revealed between 1A–1R and 1B–1R translocation forms. Thus, all of tested dh₁- and dh₂-grains of the double translocation lines showed the expected doublet: the 1A–1R translocation (Amigo)-typical rye band and the 1B–1R translocation (Kawkas)-typical rye band. Consequently, gliadin patterns estimated after SDS electrophoresis may be used as markers for the fast detection of the desired 1A–1R, 1B–1R double translocation forms among 1A–1R single translocation lines, 1B–1R single translocation lines and lines without any 1R-translocation in the progenies of appropriate crosses. Furthermore, by means of gliadin tests on the dh₂-generation the excellent stability of the double translocation 1A–1R, 1B–1R during more than one propagation phase has been proven. Estimations of high-molecular weight (HMW) glutenin subunits coded by 1A and 1B chromosomes are compatible with the double translocation constitution. A few deviating results can be explained by crossing-over events. Seed protein analysis revealed that it is possible to produce 1A–1R, 1B–1R double translocation lines with good glutenin compositions provided that adequate favourable parents are used.Former name: Department of Physiology of Institute for Cereal Research     

Development of Capsicum EST–SSR markers for species identification and in silico mapping onto the tomato genome sequence

Capsicum spp. are widely cultivated for use as vegetables and spices. The Kihara Institute for Biological Research, Yokohama City University, Japan, has stocks of approximately 800 lines of Capsicum spp. collected from various regions of Central and South America, the regions of origin for Capsicum spp. In this study, 5,751 primer pairs for simple sequence repeat markers, based on 118,060 publicly available sequences of expressed sequence tags of Capsicum annuum, were designed and subjected to a similarity search against the genomic sequence of tomato, a model Solanaceae species. Nucleotide sequences spanning 2,245 C. annuum markers were successfully mapped onto the tomato genome, and 96 of these, which spanned the entire tomato genome, were selected for further analysis. In genotyping analysis, 60 out of the 77 markers that produced specific DNA amplicons showed polymorphism among the Capsicum lines examined. On the basis of the resulting data, the 192 tested lines were grouped into five main clusters. The additional sequencing analysis of the plastid genes, matK and rbcL, divided the resources into three groups. As a result, 19 marker loci exhibited genotypes specific to species and cluster, suggesting that the DNA markers are useful for species identification. Information on the DNA markers will contribute to Capsicum genetics, genomics, and breeding.     

Value of proBNP1–108 testing for the risk stratification of patients with systolic heart failure

The study objectives were to determine the circulating levels of proBNP_1–108, the precursor of B-type natriuretic peptide (BNP) and amino-terminal pro-BNP (NT-proBNP), in patients with systolic heart failure (HF) and to assess their prognosis value for cardiovascular (CV) death over a long-term follow-up. Seventy-three patients with systolic HF and 68 healthy volunteers were included. ProBNP_1–108, BNP and NT-proBNP levels were measured with automated immunoassays and their predictive value for long-term survival was assessed through an 8 years follow-up. ProBNP_1–108 levels were markedly increased in patients with systolic HF in comparison to healthy volunteers. In univariate proportional hazard model, survival was related to proBNP_1–108, BNP, NT-proBNP, age, EF and glomerular filtration rate (eGFR). Kaplan–Meier survival curves according to proBNP tertiles diverged significantly, and the highest proBNP levels were related to patients with the highest risk of CV death. In a multivariate analysis including age, EF, proBNP_1–108, BNP, NT-proBNP, and eGFR levels, NT-proBNP was the strongest predictor of long term CV death. Our study therefore demonstrated that high levels of proBNP_1–108, measured with an assay with enhanced analytical specificity, are related to the long-term risk of cardiovascular death in systolic heart failure.     

Co-Composting of Soil Heavily Contaminated with Creosote with Cattle Manure and Vegetable Waste for the Bioremediation of Creosote–Contaminated Soil

Mispah form (FAO: Lithosol) soil contaminated with >380 000 mg kg^?1 creosote was co-composted with cattle manure and mixed vegetable waste for 19 months. The soil was mixed with wood chips to improve aeration and then mixed with cattle manure or mixed vegetable waste in a ratio of 4:1. Moisture, temperature, pH, ash content, C:N ratios, and the concentrations of creosote in the compost systems were monitored monthly. The concentrations of selected hydrocarbons in the compost systems were determined at the end of composting. Temperature rose to about 45°C in the cattle manure compost within two months of incubation while temperature in the control and vegetable waste remained below 30°C until the fourth month. Creosote concentration was reduced by 17% in the control and by more than 99% in the cattle manure and vegetable waste compost after composting. The rate of reduction in concentration in the mixed vegetable waste compost was initially lower than in the cattle manure compost. The reduction rate became similar in later months with only small differences towards the end of the composting. The concentrations of selected creosote components were reduced by between 96% and 100% after composting. There was no significant difference in reduction in concentration in both compost systems at p 0.05. Microbial activity correlated with reduction in creosote concentration.     

A new method for the quantification of superoxide dismutase mimics with an allopurinol–xanthine oxidase–lucigenin enhanced system

Allopurinol is a prodrug converted to oxypurinol by xanthine oxidase, a process followed by an efficient enzyme inhibition. Using a lucigenin-enhanced chemiluminescence method, we found that, under alkaline conditions, superoxide radicals are produced in large amounts in the first step of the interaction between the enzyme and the inhibitor. A comparison between lucigenin and cytochrome c as final detectors revealed that only the chemiluminescence technique is able to detect the superoxide anions from allopurinol oxidation. The allopurinol–xanthine oxidase–lucigenin system can be used for the quantification of various free-radical scavengers, in particular superoxide dismutase mimics. Three manganese compounds from different structural classes [manganese(II) chloride, manganese N,N′-bis(salicylidiene)ethylenediamine chloride, and manganese(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin] were compared at five concentrations (0.01, 0.1, 1, 10, and 100 μM). The method is fast, 16 times more sensitive than the cytochrome c assay at pH 10.1 and could be used for in vivo investigations avoiding the lucigenin redox cycle. If the concentrations of the reagents are increased and Tween 20 is added, the method is also operative at pH 7.4.     

Structure–Activity Studies of Brassinosteroids and the Search for Novel Analogues and Mimetics with Improved Bioactivity

A number of novel brassinosteroid analogues were synthesized and subjected to the rice leaf lamina inclination bioassay. Modified B-ring analogues included lactam, thiolactone, cyclic ether, ketone, hydroxyl, and exocyclic methylene derivatives of brassinolide. Those derivatives containing polar functional groups retained considerable bioactivity, whereas the exocyclic methylene compounds were devoid of activity. Analogues containing normal alkyl and cycloalkyl substituents at C-24 (in place of the isopropyl group of brassinolide) showed an inverse relationship between activity and chain length or ring size, respectively. The corresponding cyclopropyl and cyclobutyl derivatives were significantly more active than brassinolide and appear to be the most potent brassinosteroids reported to date. When synergized with the auxin indole-3-acetic acid (IAA), their bioactivity can be further enhanced by 1–2 orders of magnitude. The cyclopropyl derivative, when coapplied with the auxin naphthaleneacetic acid, gave a significant increase in yield of wheat in a field trial. Certain 25- and 26-hydroxy derivatives are known metabolites of brassinosteroids. All of the C-25 stereoisomers of 25-hydroxy, 26-hydroxy, and 25,26-dihydroxy derivatives of brassinolide were prepared and shown to be much less active than brassinolide. This indicates that they are likely metabolic deactivation products of the parent phytohormone. A series of methyl ethers of brassinolide was synthesized to block deactivation by glucosylation of the free hydroxyl groups. The most significant finding was that the compound where three of the four hydroxyl groups (at C-3, C-22, and C-23) had been converted to methyl ethers retained substantial bioactivity. This type of modification could, in theory, allow brassinolide or 24-epibrassinolide to resist deactivation and thus offer greater persistence in field applications. A series of nonsteroidal mimetics of brassinolide was designed and synthesized. Two of the mimetics showed significant bioactivity and one had bioactivity comparable to brassinolide, but only when formulated and coapplied with IAA. They thus represent the first nonsteroidal analogues possessing brassinosteroid activity.     

Comparison of procainamide and 2-aminobenzamide labeling for profiling and identification of glycans by liquid chromatography with fluorescence detection coupled to electrospray ionization–mass spectrometry

One of the most widely used methods for glycan analysis is fluorescent labeling of released glycans followed by hydrophilic interaction chromatography–(ultra-)high-performance liquid chromatography [HILIC–(U)HPLC]. Here, we compare the data obtained by (U)HPLC–fluorescence (FLR) coupled to electrospray ionization–mass spectrometry (ESI–MS) for procainamide and 2-aminobenzamide (2-AB)-labeled N-glycans released from human immunoglobulin G (IgG). Fluorescence profiles from procainamide show comparable chromatographic separation to those obtained for 2-AB but gave higher fluorescence intensity as well as significantly improved ESI efficiency (up to 30 times that of 2-AB). Thus, labeling with procainamide increases the ability to identify minor glycan species that may have significant biological activity.     

Abstracts of the 4th annual meeting of the European society for chronobiology jointly with the 3rd annual meeting of the British society for chronobiology,Birmingham, UK., July 9–12,1988

Abstract

In common voles (Microtus arvalis) and golden hamsters (Mesocricetus auratus) circadian changes in the susceptibility to acute dosing of rodenticides, zinc phosphide and crimidine, were investigated. Animals were dosed at four different times of day (2.00, 8.00, 14.00, 20.00 h) and mortality, convulsive seizure, latency period to first seizure, and running wheel activity were studied. Both voles and hamsters showed the highest susceptibility to zinc phosphide during the light period. In case of crimidine, circadian pattern of susceptibility was noted only in hamsters with the highest mortality during the beginning of the dark period. The data demonstrate that circadian changes in susceptibility to rodenticides have to be taken into account.